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Growth of Human Melanocyte Cultures Supported by 12-O-Tetradecanoylphorbol-13-acetate Is Mediated through Protein Kinase C Activation¹

Comprehensive Cancer Center and Institute of Cancer Research, Columbia University, New York, New York 10032 [K. R. O'D., I. B. W.], and Medical Research Division, Lederle Laboratories, American Cyanamid Company, Pearl River, New York 10965 [Y. A.]

To investigate the role of protein kinase C (PKC) in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-dependent growth of human melanocytes, we analyzed the effects of phorbol ester treatment on both PKC expression and growth control in these cells. We found that established cultures of normal melanocytes contain the PKC, PKCß, and PKC isoforms. The abilities of various phorbol ester compounds to stimulate DNA synthesis in these cultured melanocytes correlated with their known potencies for activation of PKC and tumor promotion. Dose-response studies revealed that the most effective TPA concentration for stimulation of DNA synthesis and growth of melanocytes (10 ng/ml TPA) also supported a relatively high level of PKC enzyme activity, increased membrane association of the PKC and PKC isoforms, and led to a high level of phosphorylation of a major PKC substrate, the myristoylated alanine-rich C kinase substrate (MARCKS) protein. Melanocytes incubated for 48 h with TPA at a higher concentration (100 ng/ml TPA) exhibited suboptimal TPA-stimulated DNA synthesis (28% of maximal) and decreased phosphorylation of the MARCKS substrate protein (50% of maximal). Furthermore, treatment of melanocytes with 100 ng/ml TPA for 48 h resulted in a marked decrease in total PKC enzyme activity and the loss of expression of the PKC and PKC isoforms in both the cytosol and membranebound fractions, when examined by immunoblot analysis. These results, taken together, suggest that continuous activation of PKC by TPA, rather than the loss of PKC due to TPA-induced down-regulation, is responsible for the growth-stimulatory effects of phorbol esters on normal human melanocytes. Additionally, the conditioned medium from TPA-treated human melanocytes stimulated DNA synthesis in quiescent melanocytes and human melanoma cells, thus suggesting that activation of the PKC signaling pathway in melanocytes leads to the production of an autocrine growth factor. These findings may be relevant to the autonomous growth of malignant melanomas.

¹ Funding was provided by the American Cancer Society (Grant BC-560 to M. Eisinger), the National Cancer Institute (Grant CA 087422 to M. Eisinger and Grant CA 26056 to I. B. W.), and a National Research Service Award (CA09529-05 to K. R. O'D.).

² Y. A. and K. R. O'D. contributed equally to this work and can both be considered first authors.

³ To whom requests for reprints should be addressed, at the Comprehensive Cancer Center, Columbia University, 701 West 168th St., HHSC Room 1509, New York, NY 10032.

Received 3/12/92. Accepted 6/ 4/92.

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